The present invention relates to a method and means for the determination of the hormones human chlorionic gonadotropin (HCG), luteinizing hormone (LH) and prolactin (PRL) and more especially to a fundamentally new method and means for detection of pregnancy in the human female.
Tests for the detection of pregnancy are generally based on the determination of hormones which are produced by the developing placenta, such as gonadotropic hormones similar to those produced by the anterior pituitary gland and steroid hormones similar to those of the ovary and adrenal gland. Pregnancy tests in use today are nearly exclusively based upon an assay for the placental hormone, human chorionic gonadotropin (HCG). This hormone is found in body fluids (blood serum and urine) only during pregnancy, with the exception of several other very rare hormone-producing conditions of the body. The International Unit (I.U.) of HCG was adopted in 1938 and is defined as the specific gonadotropic activity of 0.1 mg. of a dried standard kept at the National Institutes of Health, London, England.
The earliest tests for pregnancy were based upon biological in vivo methods for determining the presence of HCG. For example, the earliest test, the Aschheim-Zondek test was based upon the ability of HCG injected subcutaneously in mice to produce corpora lutea. The Friedman test is another biological test in which a urine sample of the suspected pregnancy is injected into the ear vein of a mature female rabbit which has been isolated 3 to 4 weeks, and 48 hours after injection the ovaries are examined for ruptured hemorrhagicfollicles, which indicate a positive reaction. A lesser known test developed by Kupperman in 1943 involves the injection of the patient's urine into a female rat with subsequent inspection of the ovary for signs of hyperemia. While the two hours necessary for conducting this test is considerably shorter than the 48 hours required for the Friedman test and the nearly five days required for the Aschheim-Zondek test, this test is not as reliable since it requires a skilled technician to differentiate a slightly pink negative ovary from a reddened positive ovary to attain a high degree of accuracy. A test developed by Galli and Mainini in the late 1940's also requires only approximately two hours to conduct the test; however, this test involves the injection of the patient's urine into frogs with the subsequent observation for ejection of sperms, and these animals are relatively insensitive compared with rabbits, mice and rats.
All of the foregoing biological test suffer from serious disadvantages, including the availability of animals, the need to maintain a large colony of animals, and relatively long periods of testing, frequent yielding of false positive and negative results, and most significantly, the fact that a positive test can be achieved with only a 95% degree of reliability only after a period of 25 to 30 days following ovulation.
A second generation of pregnancy tests developed during the early 1960's are characterized as immunological or immunochemical procedures. Since HCG is a protein hormone, it acts antigenically in a heterologous species. Accordingly, when HCG is injected by suitable techniques into an appropriate test animal, most typically a rabbit, an antibody to HCG is produced within the animal. In early tests utilizing this principle, it was attempted to utilize the antigen-antibody direct precipitin reaction, according to which a visible precipitate would form as a result of the combination of HCG and its antibody, to detect the presence of this hormone. More common procedures involved so-called indirect methods of determination, such as the latex particle slide test of Brody and Carlstrom and hemagglutination and inhibition test of Wide and Gemzel. In the former procedure, an antiserum is added to the urine of a patient followed by a latex carrier which has been coated with HCG. If the urine specimen is of a pregnant woman and contains HCG, the antibodies in the antiserum will be neutralized and will not therefore react with the HCG coated on the carrier to produce agglutination. In the latter testing method, a similar principle is employed except that the indicator comprises HCG conjugated with red blood cells or formalinized red blood cells. The first test may be carried out in a period of only about two minutes whereas the second requires approximately two hours. Either the patient's urine or blood may be employed for these test procedures.
In a very recently developed pregnancy testing procedure based upon a modification of the basic immunological mechanism, radiological means are employed to detect and/or measure the presence of HCG in the patient's blood (or urine). See, for example, Goldstein et al. in Fert. Steril., Vol. 23, page 817 (1972). In these radioimmunoassay tests, the antibody is placed in contact with a mixture of the patient's body fluid to be tested and a known amount of HCG tagged with a radioactive isotope, and the HCG in the test sample and the labeled HCG compete for interaction with the HCG antibody. The antibody is then separated from the fluid and either fraction may be analyzed radiologically to determine the respective proportions of the labeled and unlabeled HCG which became bound to the antibodies, and the concentration of HCG in the sample can be calculated from this information since the proportion of labeled and unlabeled HCG will be in the same proportion in both fractions. The radioimmunoassay techniques have overcome one significant limitation of the immunochemical pregnancy test, namely, that of sensitivity. Radioimmunoassay techniques are several thousand times more sensitive than the above-described indirect tests, and accordingly, permit a detection of pregnancy much earlier than the 25 to 30 days following ovulation required with the latex particle slide test and the hemagglutination test. However, even though pregnancy may be detected after the 10th or 12th day following ovulation with a 95% degree of reliability by the radioimmunoassay method, these tests have the disadvantage that they require more time to carry out, typically about 24-48 hours.
The most serious drawback, however, with all previously known pregnancy testing techniques involves the frequent indication of false positive and negative results. In the case of the immunochemical pregnancy test, this difficulty is due to non-specific immune response related to non-specific antibody-antigen reactions. For example, the presence of a common hormone, non-specific alpha-subunit among follicle stimulating hormone (FSH), luteinizing hormone (LH), HCG and thyroid stimulating hormone (TSH), and homologies in the amino acid sequence of the hormone-specific beta-subunits have caused further difficulties in producing specific antisera for use in the immunochemical techniques. These difficulties are exaggerated in radioimmunoassay methods due to the high degree of sensitivity of this technique. This last-mentioned drawback has been partially circumvented by producing antisera specific to the beta-subunit of HCG; however, this manipulation requires the use of valuable material, immunization, selection and purification to achieve specific antibodies, which procedures are time consuming, expensive and very cumbersome. In spite of these precautions, the goal of near 100% reliability in the detection of pregnancy by radioimmunoassay has not been accomplished.
Furthermore, a serious need exists for a means to detect ectopic pregnancies at the earliest possible moment. Such pregnancies have negative hemagglutination or latex slide tests in 40 to 60% of all patients, even after the 25-30 day period following ovulation required to perform these tests. The reason for this relates to the belief that the immunochemical pregnancy testing techniques provide for detection of a pregnancy only after implantation of the fertilized ovum has occurred. As described hereinabove, all of the heretofore known pregnancy testing methods suffer from this disadvantage in that they are not effective until after nidation. A test which could determine the presence of a pregnancy during the period between fertilization of the ovum and implantation of the fertilized ovum would be invaluable in the treatment of, for example, threatened abortion in women who habitually abort, cases involving artificial insemination and in connection with new contraceptive methods which effectively terminate a pregnancy prior to implantation.
The presence of specific receptors for the various hormones has long been suspected in target cases of both humans and animals, and researchers have had success within the last half decade in identifying and directly studying certain of these receptors and their interaction with many respective hormones. Preliminary studies undertaken by the present inventor have suggested the presence of a receptor for HCG in the corpus luteum of pregnant or pseudo-pregnant rats. Gonadotropins, Chapter 21, 1972, John Wiley & Sons. These preliminary studies, however, gave no indication of whether a similar receptor is present in humans or other animals, whether the receptor found in rats is species specific or specific even for HCG, whether it would be a reliable indicator for HCG in body fluid samples or whether it is stable over any extended period of time.
Prolactin (PRL) represents a hormone which for many years was not believed to be present in humans. Its presence in humans was confirmed in the early 1970's. The presence of PRL receptors in mammary tissue has been documented, and the hormone is believed to play a role in breast cancer, pituitary tumors, lactation disorders, hypothyroidism and other disorders. There are thought to be as many as 84 different functions of PRL involving complex interaction with other pituitary gonadal and adrenal hormones, possibly in the role as an intermediary regulator for these other hormones. A luteotropic as well as a luteolytic role of PRL during the estres cycle of the rat has also been suggested, and there is some evidence developed in the last few years regarding studies of the rat. The specific binding of PRL to rat ovary has been suggested very recently in the literature, but no specific receptor has been identified. Turkington et al., Rec. Prog. Horm. Res., 29:417 (1973). Studies by several investigators have suggested that PRL has little function in the maintenance of corpus luteum in humans. Hwang et al., Proc. Nat. Acad. Sci., 68:1902 (1971); Midgley et al., Proc. IV Int. Cong. Endocrinal, Int. Cong. Series No. 273, Excerpta Medica, Amsterdam, 1972.
In view of the maladies identified above which are known to involve the hormone PRL, it is desired oftentimes to determine and/or monitor the PRL content in a test sample obtained from a subject, for example, a sample of body fluid. In some cases, only small samples are available, e.g., pediatric cases, and therefore, it is desired to have a very specific test which requires only a small sample and only a short period of time to carry out. Specificity is desired in order to differentiate from other hormone species which may be present in the sample, and this is particularly true in cases where it might be desired simultaneously determine or measure two or more different hormones. There exists at present no truly specific means for making such a determination.